Method and apparatus for detecting cancer, influenza, or HIV based on α-N-acetyl-galactosaminidase detection

ABSTRACT

HIV- and influenza virus-infected cells secrete α-N-acetylgalactosaminidase into the blood stream. Secretion of α-N-acetylgalactosaminidase is also associated with cancer. Elevated levels of α-N-acetylgalactosaminidase in the blood stream result in the deglycosylation of the Gc protein. This inactivates the MAF precursor activity of the Gc protein, leading to immunosuppression. Thus, the α-N-acetylgalactosaminidase activity in a patient&#39;s bloodstream can serve as a diagnostic and prognostic index. Antibody-sandwich ELISA methods and kits for α-N-acetylgalactosaminidase as an antigen were developed to detect serum or plasma α-N-acetylgalactosaminidase activity in patients and are used as a diagnostic/prognostic index.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/039,159,filed Mar. 13, 1998, which is a continuation of application Ser. No.08/938,553, filed Sep. 26, 1997, now U.S. Pat. No. 5,776,671 which is acontinuation-in-part application of Ser. No. 08/779,729, filed Jan. 6,1997, now U.S. Pat. No. 5,712,104 and application Ser. No. 08/618,485filed Mar. 19, 1996, the entire disclosures of which are incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates to diagnostic and prognostic methods utilizing ANenzyme-linked immunosorbent assay (ELISA) to detect the specific enzyme,α-N-acetylgalactosaminidase that causes immunosuppression, found in theblood stream of AIDS/HIV-infected and influenza patients.

    ______________________________________                                        TABLE OF TERMS                                                                ______________________________________                                        Gc protein                                                                              Vitamin D.sub.3 -binding protein                                      MAF macrophage activating factor                                              GcMAF Gc protein-derived macrophage activating factor                         PBMC peripheral blood mononuclear cells                                       HA hemagglutinin                                                              Nag α-N-acetylgalactosaminidase                                         NagAg α-N-acetylgalactosaminidase as an antigen                         ELISA enzyme-linked immunosorbent assay                                     ______________________________________                                    

SUMMARY OF THE INVENTION

Vitamin D₃ -binding protein (Gc protein) is the precursor for macrophageactivating factor (MAF). HIV- and influenza virus-infected cells secreteα-N-acetylgalactosaminidase into the blood stream, resulting in thedeglycosylation of Gc protein. This inactivates the MAF precursoractivity of the Gc protein, leading to immunosuppression. Thus,α-N-acetylgalatosaminidase activity in a patient's blood stream canserve as a diagnostic and prognostic index. An antibody-sandwich ELISAmethod and kits for HIV and influenza specificα-N-acetylgalactosaminidase as antigens were developed to detect serumor plasma α-N-acetylgalactosaminidase in AIDS/HIV-infected and influenzapatients and used as a diagnostic/prognostic index.

DESCRIPTION OF THE DRAWINGS

Other objects and many attendant features of this invention will becomereadily appreciated as the same becomes better understood by referenceto the following detailed description when considered in connection withthe accompanying drawings wherein:

FIG. 1A is a schematic illustration of the conversion of Gc protein tomacrophage activating factor (MAF).

FIG. 1B is a schematic illustration of the deglycosylation of Gc proteinin HIV- or influenza-infected patient's blood stream.

FIG. 2 shows the standard correlation betweenα-N-acetylgalactosaminidase as a HIV-antigen and absorbance (colordensity) for alkaline phosphatase activity of ELISA.

FIG. 3 shows the standard correlation betweenα-N-acetylgalactosaminidase as an influenza-antigen and absorbance(color density) for alkaline phosphatase activity of ELISA.

BACKGROUND OF THE INVENTION

A. Immunosuppression Resulted from Loss of MAF Precursor Activity

Infections result in inflammation which attracts and activatesmacrophages. Inflamed lesions release lysophospholipids. Administrationinto mice of small doses (5-20 μg/mouse) of lysophosphatidylcholine(lyso-Pc) and other lysophospholipids resulted in the greatly enhancedphagocytic and superoxide generating capacities of macrophages (Ngwenyaand Yamamoto, Proc. Soc. Exp. Biol. Med. 193:118, 1990; Yamamoto et al.,Inf. Imm. 61:5388, 1993; Yamamoto et al., Inflammation. 18:311, 1994).The inflammation-primed macrophage activation process is the majormacrophage activation cascade (Yamamoto, Mol. Immunol. 33:1157, 1996),which requires the participation of B and T lymphocytes and serumvitamin D binding protein (DBP; human DBP is known as group specificcomponents or Gc protein). The inducible β-galactosidase (Bgl_(i)) ofinflammation-primed (or lyso-Pc-treated) B cells and the Neu-1 sialidaseof T cells convert Gc glycoprotein to the macrophage activating factor(MAF), a protein with N-acetylgalactosamine as the remaining sugarmoiety (FIG. 1a) (Yamamoto and Homma, Pro. Natl. Acad. Sci. USA.88:8539, 1991; Yamamoto, Mol. Immunol. 33:1157, 1996). Thus, the Gcprotein is a precursor for MAF. Incubation of Gc protein withimmobilized β-galactosidase and sialidase generates a remarkably hightitered MAF (termed GcMAF) (Yamamoto, Mol. Immunol. 33:1157, 1996;Yamamoto and Kumashiro, J. Immunol. 151:2794, 1993; Naraparaju andYamamoto, Immunol. Lett. 43:143, 1994; U.S. Pat. Nos. 5,177,002 and5,326,749). Administration of a minute amount (10 pg/mouse; 100ng/human) of GcMAF resulted in greatly enhanced phagocytic andsuperoxide generating capacities of macrophages.

When peripheral blood monocytes/macrophages (phagocytes) of over 250HIV-infected patients were treated with a small amount (100 pg/ml) ofGcMAF, the phagocytes of all patients were activated for generating morethan 4.0 nmoles of superoxide/min/10⁶ phagocytes as with healthy humans.When a mixture of lymphocytes and phagocytes was treated with 1 μglyso-Pc/ml for 30 minutes and cultured in a medium supplemented with Gcprotein (1 ng/ml) for 3 h, the phagocytes of all patients were activatedfor generating more than 4.0 nmoles of superoxide/min/10⁶ phagocytes.The results indicate that lymphocytes of these patients are capable ofconverting Gc protein to MAF. However, when a mixture of lyso-Pc-treatedlymphocytes and phagocytes was cultured in a medium supplemented withpatient own serum (0.1%) as a source of Gc protein for 3 hours, thephagocytes were not activated with patient serum of about 10% of thispatient population and produced less than 0.7 nmoles ofsuperoxide/min/10⁶ phagocytes (Yamamoto et al., AIDS Res. Human Retro.11:1373, 1995). These patients having severely decreased precursoractivity of serum Gc protein were found to be approximately 1/4 of theAIDS patients. However, electrophoretic analysis showed no detectablechange in either quantity or molecular weight of plasma Gc protein.Thus, the lost or reduced precursor activity of a patient's Gc proteinis due to the deglycosylation of the Gc protein (Yamamoto et al., AIDSRes. Human Retro. 11:1373, 1995). Deglycosylated Gc protein cannot beconverted to MAF (FIG. 1b). Therefore, macrophage activation cannotdevelop in certain AIDS/HIV-infected patients. Since macrophageactivation for phagocytosis and antigen presentation is the first stepin the immune development cascade, the patients incapable of macrophageactivation become severely immunosuppressed. This may explain why AIDSpatients die from overwhelming opportunistic infection. The MAFprecursor activity of Gc protein in the sera of approximately 25% ofthis patient population was moderately reduced (ranging from 1.6-3.6nmoles of superoxide produced/min/10⁶ phagocytes). The remainingasymptomatic (65%) HIV-infected patients had MAF precursor activitiessimilar to those of healthy humans.

All HIV-infected patient's sera were found to containα-N-acetylgalactosaminidase that deglycosylates Gc protein (Yamamoto etal., AIDS Res. Human Retro. 11:1373, 1995). Patients having severelydecreased precursor activity carry high serumα-N-acetylgalactosaminidase activity whereas the asymptomatic patientshaving high precursor activity carry a very low level of serumα-N-acetylgalactosaminidase activity. Thus, plasmaα-N-acetylgalactosaminidase activity has an inverse correlation with theMAF precursor activity of Gc protein (Yamamoto et al., AIDS Res. HumanRetro. 11:1373, 1995).

Similarly, an influenza patient's precursor activity is lost ordecreased because serum Gc protein is deglycosylated byα-N-acetylgalactosaminidase found in the blood stream of influenzapatients. Since the deglycosylated Gc protein cannot be converted toMAF, the lack of macrophage activation in influenza patients leads toimmunosuppression, which causes the secondary bacterial infections(i.e., pneumonia).

In my prior three U.S. Pat. Nos. 5,177,002, 5,326,749, and 5,620,846,the entire disclosures of which are incorporated by reference herein, asare my above cited journal articles, is disclosed the macrophageactivating factor, processes for preparing them as well as methods ofinducing macrophage activation in a person in need of such activation,and diagnostic or prognostic assays of serum α-N-acetylgalactosaminidasein cancer and AIDS patients.

B. The Origin of α-N-acetylgalactosaminidase

Serum α-N-acetylgalactosaminidase activity was found in the blood streamof all stages of HIV-infected patients but not in healthy humans(Yamamoto et al., AIDS Res. Human Retro. 11:1373, 1995). In contrast,serum β-N-acetylglucosaminidase activity levels of healthy humans areequivalent to those of HIV-infected patients. Thus, serumβ-N-acetylglucosaminidase seems to be clinically insignificant. In fact,α-N-acetylgalactosaminidase activity is responsible for thedeglycosylation of Gc protein because the Gc protein is O-glycosylated(Yamamoto et al., AIDS Res. Human Retro. 11:1373,1995).

The deglycosylation of Gc protein by serum α-N-acetylgalactosaminidaseappears to be the major cause for immunosuppression in AIDS/HIV-infectedpatients. Cultured HIV-infected cells can secreteα-N-acetylgalactosaminidase into culture medium. When peripheral bloodmononuclear cells (PBMC) of HIV-infected patients were cultured for 3days, small amounts of α-N-acetylgalactosaminidase were detected in theculture media. If these PBMC were treated with mitomycin C (5 μg/ml) orBUDR as a provirus inducer (Sato et al., Arch. Virol. 54:333, 1977) for30 min and cultured for 3 days, α-N-acetylgalactosaminidase enzymeactivity in culture media increased significantly. This enzyme isimmunoprecipitable with polyclonal anti-HIV antibody, indicating thatthis enzyme is a viral gene product. Moreover, serumα-N-acetylgalactosaminidase of HIV-infected patients wasimmunoprecipitable without addition of anti-HIV antibody, suggestingthat the enzyme is already complexed with patient own immunoglobulin G.The immunogenicity of the serum enzyme also suggests that the serumenzyme is likely to be coded by a viral gene. In fact, the HIV-envelopeprotein, gp120, was found to contain α-N-acetylgalactosaminidaseactivity.

Similarly, all influenza infected patient's sera containα-N-acetylgalactosaminidase. An influenza virus envelope protein,hemagglutinin (HA), was found to contain α-N-acetylgalactosaminidaseactivity. Influenza virus-infected cells can secrete this enzyme asunassembled HA protein and its fragments into the blood stream,resulting in the deglycosylation of Gc protein. The deglycosylated Gcprotein cannot be converted to MAF. Thus, lack of macrophage activationin influenza patients leads to immunosuppression, which frequentlycauses the secondary bacterial infections (i.e., pneumonia). Since theserum α-N-acetylgalactosaminidase of influenza patients was found to bea viral coded product and resides on the HA protein, theα-N-acetylgalactosaminidase activity in a patient's bloodstream appearsto be indicative of virus load and can serve as a diagnostic andprognostic index for these influenza patients.

All viral components in HIV and influenza are antigenically distinct,serum α-N-acetylgalactosaminidase activities as thediagnostic/prognostic indices of these patients are antigenicallydistinguishable among these individual viral diseases. Thus, ELISA forthese viral enzymes should be valuable for diagnosis and prognosis ofthese diseases.

C. Diagnostic and Prognostic Significance of α-N-AcetylgalactosaminidaseActivity in Blood Stream of AIDS/HIV-infected and Influenza Patients

1. Assay Protocol for Detection of α-N-acetylgalactosaminidase in BloodStream of AIDS/HIV-infected and Influenza Patients

i) Schematic Illustration.

Detection procedure for deglycosylating enzyme of serum Gc protein,α-N-acetylgalactosaminidase, in AIDS/HIV-infected and influenzapatient's blood stream.

Step. I. Stepwise 30 and 70% ammonium sulfate precipitation of plasma orserum:

Serum/plasma (1 ml)+30% and then 70% saturated ammonium sulfate 70%precipitate→dissolved in 50 mM citrate phosphate buffer (pH6.0)→dialyzed against the same buffer at 4° C. for overnight.

The dialysates was made up to 1 ml in volume.

Step. II. Enzyme assay of α-N-acetylgalactosaminidase

Reaction mixture: 250 μl of the dialyzed sample+250 ml of 50 mM citratephosphate buffer (pH 6.0) containing 5 μmoles of p-nitrophenylN-acetyl-α-D-galactosaminide as substrate.

Incubation time: 60 min, terminated by adding 200 μl of 10% TCA. Aftercentrifugation of the reaction mixture, 300 μl of 0.5 M Na₂ CO₃ wasadded to the supernatant.

Activity measurement: absorbance of amount of released p-nitrophenol wasdetermined spectrophotometrically at 420 nm with Beckman DU 600 Spectrophotometer and expressed as specific activity unit of nmole/mg/min.Protein concentrations were determined by the Bradford method.

ii) Descriptive Enzyme Assay Procedure for α-N-acetylgalactosaminidase.

Plasma/serum (1 ml) of patients was precipitated with 70% saturatedammonium sulfate. The ammonium sulfate precipitate was dissolved in 50mM citrate phosphate buffer (pH 6.0) and dialyzed against the samebuffer at 4° C. The volume of the dialysate was made up to 1 ml.Ammonium sulfate precipitation also separates the enzyme from productinhibitors. The substrate solution (250 μl) contained 5 μmoles ofp-nitrophenyl N-acetyl-α-D-galactosaminide in a 50 mM citrate phosphatebuffer (pH 6.0). The reaction was initiated by addition of 250 μl of thedialyzed samples, kept at 37° C. for 60 min and terminated by adding 200μl of 10% TCA. After centrifugation of the reaction mixture, 300 μl of0.5 M Na₂ CO₃ solution was added to the supernatant. The amount ofreleased p-nitrophenol was determined spectrophotometrically at 420 nmwith a Beckman DU 600 Spectrophotometer and expressed as specificactivity unit of nmole/mg/min. Protein concentration were estimated byBradford method (Bradford, Anal Biochem 72: 248, 1976).

2. Diagnostic/Prognostic Utility of Serum α-N-acetylgalactosaminidasefor Patient Infected with HIV or Influenza.

Serum α-N-acetylgalactosaminidase activity assesses infected viral loadbecause the serum enzyme level is proportional to the total amount ofunassembled viral envelope components (i.e., gp120 for HIV and HA forinfluenza), which is indicative of viral load.

a) HIV Infection.

As shown in Table 1, serum α-N-acetylgalactosaminidase activity of thefirst 14 patients out of 250 patients represents a diagnostic indexwhich shows inverse correlation with the MAF precursor activity of Gcprotein but no obvious correlation with CD4⁺ values (Yamamoto et al.,AIDS Res. Human Retro. 11:1373, 1995).

b) Influenza Virus Infection.

As shown in Table 2, serum α-N-acetylgalactosaminidase activity of 12influenza patients represents diagnostic index that shows an inversecorrelation with the MAF precursor activity of their serum Gc protein.

DESCRIPTION OF THE INVENTION DESCRIPTION OF THE ENZYME-LINKEDIMMUNOSORBENT (ELISA) ASSAY PROCEDURE FOR α-N-ACETYLGALACTOSAMINIDASEACTIVITY AS AN ANTIGEN IN BLOOD STREAM OF AIDS/HIV-INFECTED ANDINFLUENZA PATIENTS

In the immunoassay procedure for the detection of serum or plasmaα-N-acetylgalactosaminidase (Nag) as an antigen (NagAg),antibody-sandwich ELISA kits were prepared.

1. Preparation of antibody. The viral Nag enzyme was purified from seraof HIV-infected patients or embryonated chicken egg grown influenzavirus-lysates (excluding complete virions) and used for immunization ofanimals (rabbit, goat and mouse). Polyclonal antibodies (goat andrabbit) and monoclonal antibodies against α-N-acetylgalactosaminidase(NagAg) were prepared. An immunoglobulin fraction was purified fromantisera or monoclonal ascites fluid using ammonium sulfate (50%saturated) fractionation and DE52 ionic exchange column with 10 mM PBS(0.15 M NaCl and 10 mM phosphate buffer) or protein A columns.

2. Conjugation of alkaline phosphatase to antibodies. Dialyze 5 mg/mlmonoclonal or polyclonal antibody solution in 0.1 M phosphate buffer atpH 6.8 (PBS) overnight at 40° C. Add 0.5 mg of dialyzed antibody to 1.5mg of alkaline phosphatase (immunoassay grade; Boehringer Mannheim,Indianapolis, Ind.) in 10 ml of 10 mM PBS. Add 80 μl 25% glutaraldehydeand mix gently. Let stand at room temperature for 2 hrs. Stop reactionby adding an equivalent volume (10 ml) of PBSLE (10 mM PBS containing100 mM lysine and 100 mM ethanolamine). Desalt with Sephadex G25 columnin PBSN (10 mM PBS with 0.05 M NaN₃). Mix 20 ml of alkalinephosphatase-antibody conjugates with 40 ml of blocking buffer (0.17 Mborate buffer containing 2.5 mM MgCl₂, 0.05% Tween 20, 1 mM EDTA, 0.25%BSA and 0.05% NaN₃). Filter 60 ml of the conjugates through alow-protein binding filter, Millex HV 0.45 μm (Millipore Corp. Bedford,Mass.), for sterilization and store at 4° C.

3. Preparation of antibody coated microtiter plates. Using multichannelpipets arid tips, dispense 50 μl of polyclonal or monoclonal antibodysolution 2 μg/ml in PBSN into each well of a microtiter plate(microwell). Wrap the plates in plastic wrap to seal and incubate 2 hrsat 37° C. or overnight at room temperature. Rinse the antibody coatedplate by flooding with distilled water more than three times. Fill eachwell with blocking buffer dispensed as a stream from a bottle andincubate 30 min at room temperature. Rinse the plate three times withdistilled water and remove any residual liquid by gently flicking itface down onto paper towels.

4. Antigen (NagAg) Preparation for ELISA.

a) Standard antigen for standard curve. Prepare a standardantigen-dilution series by successive dilutions of NagAg stock (e.g.,the enzyme purified from AIDS patient sera or embryonated chickenegg-grown influenza virus lysates, excluding complete virions) inblocking buffer. The activity range of Nag enzyme dilution spans from 2to 10 nmole/min, corresponding to NagAg antigens dilution ranging from12 to 60 μg/ml.

b) Test sample. Serum or plasma of HIV-infected and influenza patients.

5. ELISA.

Step I. Add 50-μl aliquots of 1/10 serum dilution in PBSN of the testNagAg sample solutions (patient serum or plasma enzyme) or the standardNagAg (enzyme) dilutions to the antibody coated wells and incubate 2 hrsat room temperature. Rinse plate three times in distilled water. Filleach well with blocking buffer and incubate 10 min at room temperature.Rinse three times with water and remove residual liquid.

Step II. Add 50 μl specific antibody-alkaline phosphatase conjugate (300ng/ml and incubate 2 hrs at room temperature. Wash plate as in Step I.

Step III. Add 75 μl of p-nitrophenyl phosphate (NPP) substrate solution(4 μmoles/ml) to each well and incubate for 1 hr at room temperature.Alkaline phosphatase activity causes the solution to change color. Colordensity (absorbance) relates to the amount of NagAg. Read the plate on amicrotiter plate reader with a 405-nm filter.

6. Data analysis. Standard curves were constructed for the ELISAproduced by the dilutions of the standard antigen (NagAg) of purifiedHIV- and influenza-enzymes. The NagAg concentrations were expressed asNag enzyme activities and plotted on the x axis and absorbance (colordensity) of alkaline phosphatase activity on the y axis, as shown inFIG. 2 for HIV and FIG. 3 for Influenza virus. Interpolate theabsorbance of the test patient serum/plasma sample to determineα-N-acetylgalactosaminidase activity. Table 3 exemplifies the patientenzyme activities determined from ELISA for sera of 16 AIDS/HIV-infectedpatients. Similar results were also observed with 14 influenza patientsas shown in Table 4.

Since antibodies to HIV and influenza virus enzymes are specific to therespective enzymes (NagAg) and do not cross-react, this antibodysandwich ELISA distinguishes individual enzymes. Thus, the presentmethod is particularly useful if these patients are dually Infected(e.g., influenza virus Infection of AIDS/HIV-infected patients).

                  TABLE 1                                                         ______________________________________                                        Serum α-N-acetylgalactosaminidase activity detected in                    HIV-infected patient plasma and its correlation with the                      precursor activity of plasma Gc protein and the CD4.sup.+  value.           N-acetylgalactosaminidase                                                                       Patient Specific activity Disease stage indices             No.   (nmoles/mg/min)  Precursor activity                                                                        CD4.sup.+  value                           ______________________________________                                        1     13.12            0.54        188                                          2 2.51 3.42 102                                                               3 12.80 0.69 136                                                              4 1.43 4.43 577                                                               5 0.51 5.14 160                                                               6 0.54 5.22 222                                                               7 1.01 4.52 298                                                               8 0.81 5.03 156                                                               9 2.63 3.14 849                                                               10 3.15 2.91 22                                                               11 2.28 3.62 585                                                              12 3.03 2.91 845                                                              13 3.54 1.64 326                                                              14 1.35 4.63 305                                                              Control.sup.a 0.24.sup.b 5.10 --                                            ______________________________________                                         .sup.a Average of 5 healthy humans.                                           .sup.b This enzyme activity in healthy humans was found to be                 galactosidase.                                                           

                  TABLE 2                                                         ______________________________________                                        Precursor activity of Gc protein and α-N-acetylgalactosaminidase         activities detected in blood stream of acute influenza patients.                        α-N-acetylgalactosaminidase                                                                Precursor activity                                Patient Specific activity Superoxide produced                                 (No) (nmole/min/mg) (nmole/min/10.sup.6  cells)                             ______________________________________                                        1      1.96               3.01                                                  2 1.75 3.27                                                                   3 4.11 1.95                                                                   4 1.68 3.45                                                                   5 1.53 4.89                                                                   6 0.87 4.39                                                                   7 2.23 2.54                                                                   8 5.93 1.26                                                                   9 3.65 2.17                                                                   10 2.23 2.65                                                                  11 1.68 4.70                                                                  12 2.83 2.33                                                                  Control 0.29.sup.a 5.86                                                     ______________________________________                                         .sup.a This enzyme activity in healthy humans was found to be                 galactosidase.                                                           

                  TABLE 3                                                         ______________________________________                                        Serum α-N-acetylgalactosaminidase activity of AIDS/HIV-infected          patients determined from ELISA.                                                Patient    Absorbance α-N-acetylgalactoeaminidase                                              No (Color density) activity (nmole/mg/min)           ______________________________________                                        1        0.325      2.37                                                        2 0.498 3.64                                                                  3 0.996 7.28                                                                  4 0.238 1.74                                                                  5 0.429 3.13                                                                  6 1.213 8.87                                                                  7 0.592 5.32                                                                  8 1.370 10.01                                                                 9 0.254 1.86                                                                  10 0.587 4.29                                                                 11 0.272 1.98                                                                 12 0.622 4.55                                                                 13 0.712 3.20                                                                 14 0.232 1.69                                                                 15 0.281 2.05                                                                 16 0.185 1.35                                                               ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Serum α-N-acetylgalactosaminidase activity of influenza                   virus-infected patients determined from ELISA.                                Patient     Absorbance α-N-acetylgalactoeaminidase                      No (Color density) activity (nmole/mg/min)                                  ______________________________________                                        1         0.188      1.64                                                       2 0.455 3.97                                                                  3 0.172 1.50                                                                  4 0.530 4.63                                                                  5 0.222 1.94                                                                  6 0.324 2.83                                                                  7 0.203 1.77                                                                  8 0.215 1.88                                                                  9 0.418 3.65                                                                  10 0.242 2.11                                                                 11 0.318 2.78                                                                 12 0.225 1.96                                                                 13 0.198 1.73                                                                 14 0.218 1.85                                                               ______________________________________                                    

References Cited

The following references are cited and their entire text is incorporatedfully herein as are all references set forth above in the specification.

U.S. PATENT DOCUMENTS

U.S. Pat. Nos. 5,177,002, 5,326,749 and 5,620,846 (Yamamoto).

OTHER PUBLICATIONS

1. Bradford, M. M., A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochem. 72: 248-254 (1976).

2. Sato, M., Tanaka, H., Yamada, T. and Yamamoto, N., Persistentinfection of BHK/WI-2 cells with rubella virus and characterization ofrubella variants. Arch. Virology 54:333-343 (1977).

3. Ngwenya, B. Z. and Yamamoto, N., Contribution oflysophosphatidylcholine treated nonadherent cells to mechanism ofmacrophage stimulation. Proc. Soc. Exp. Biol. Med. 193:118-124 (1990).

4. Yamamoto, N. and Homma, S., Vitamin D₃ binding protein(group-specific component, Gc) is a precursor for the macrophageactivating signal from lysophosphatidylcholine-treated lymphocytes.Proc. Natl. Acad. Sci. USA. 88:8539-8543 (1991).

5. Yamamoto, N. and Kumashiro, R., Conversion of vitamin D₃ bindingprotein (Group-specific component) to a macrophage activating factor bystepwise action of β-galactosidase of B cells and sialidase of T cells.J. Immunol. 151:27-94-2902 (1993).

6. Yamamoto, N., Kumashiro, R., Yamamoto, M., Willett, N. P. andLindsay, D. D., Regulation of inflammation-primed activation ofmacrophages by two serum factors, vitamin D₃ -binding protein andalbumin. Inf. Imm. 61:5388-5391 (1993).

7. Yamamoto, N., Willett, N. P. and Lindsay, D. D., Participation ofserum proteins in the inflammation-primed activation of macrophages.Inflammation. 18:311-322 (1994).

8. Naraparaju, V. R. and Yamamoto, N., Roles of β-galactosidase of Blymphocytes and sialidase of T lymphocytes in inflammation-primedactivation of macrophages. Immunol. Lett. 43:143-148 (1994).

9. Yamamoto, N., Naraparaju, V. R. and Srinivasula, S. M., Structuralmodification of serum vitamin D₃ -binding protein and immunosuppressionin HIV-infected patients. AIDS Res. Human Ret. 11:1373-1378 (1995).

10. Yamamoto N. Structural definition of a potent macrophage activatingfactor derived from vitamin D₃ binding protein with adjuvant activityfor antibody production. Molecular Immunol. 33:1157-1164 (1996).

I claim:
 1. A method for detecting whether a patient suffers from adisease selected from the group consisting of cancer, influenza and HIV,said method comprising:obtaining a plasma or serum sample from saidpatient; detecting an amount of α-N-acetylgalactosaminidase in saidsample; comparing said detected amount of α-N-acetylgalactosaminidasewith a reference amount of α-N-acetylgalactosaminidase to detect whethersaid patient suffers from said disease.
 2. The method of claim 1,wherein said disease is cancer and said α-N-acetylgalactosaminidase isspecifically associated with cancer.
 3. The method of claim 1, whereinsaid disease is influenza and said α-N-acetylgalactosaminidase isspecifically associated with influenza.
 4. The method of claim 1,wherein said disease is HIV and said α-N-acetylgalactosaminidase isspecifically associated with HIV.
 5. The method of claim 1, wherein saiddisease is detected when said detected amount ofα-N-acetylgalactosaminidase exceeds said reference amount ofα-N-acetylgalactosaminidase.
 6. A kit adapted to perform the method ofclaim 1, said kit comprising:polyclonal or monoclonal antibodiesspecific to α-N-acetylgalactosaminidase; and α-N-acetylgalactosaminidaseas an antigen standard.